Carboxyl Methyltransferase Catalysed Formation of Mono‐ and Dimethyl Esters under Aqueous Conditions: Application in Cascade Biocatalysis

Abstract Carboxyl methyltransferase (CMT) enzymes catalyse the biomethylation of carboxylic acids under aqueous conditions and have potential for use in synthetic enzyme cascades. Herein we report that the enzyme FtpM from Aspergillus fumigatus can methylate a broad range of aromatic mono‐ and dicarboxylic acids in good to excellent conversions. The enzyme shows high regioselectivity on its natural substrate fumaryl‐l‐tyrosine, trans, trans‐muconic acid and a number of the dicarboxylic acids tested. Dicarboxylic acids are generally better substrates than monocarboxylic acids, although some substituents are able to compensate for the absence of a second acid group. For dicarboxylic acids, the second methylation shows strong pH dependency with an optimum at pH 5.5–6. Potential for application in industrial biotechnology was demonstrated in a cascade for the production of a bioplastics precursor (FDME) from bioderived 5‐hydroxymethylfurfural (HMF).

Codon optimised gene was synthesised by GeneArt using the sequence from UniProt accession code Q4WZ45. This was subcloned into a pET-based golden gate acceptor vector with a C-terminally coded histidine tag and a T7 inducible promoter.

N-tagged FtpM
Codon optimised gene was synthesised by GeneArt using the sequence from UniProt accession code Q4WZ45. This was subcloned into a pET-based golden gate acceptor vector with a N-terminally coded histidine tag and a T7 inducible promoter.
pDK50 FtpM Construct used in the Kalb et al. paper that was provided by the author for this research [1] .

SAH-nucleosidase (EC 3.2.2.9)
Codon optimised gene was synthesised by GeneArt using the sequence from UniProt accession code P0AF12. This was subcloned into a pET-based golden gate acceptor vector with a C-terminally coded histidine tag and a T7 inducible promoter.

PaoABC
The plasmid pMN100 derived from pTrcHisA (Invitrogen), containing the PaoABC subunit genes with a N-terminally coded histidine tag fused to the PaoA subunit. Provided by S. Leimkuhler, University of Potsdam [2] .

Protein expression and purification
C-and N-His tagged constructs were synthesised (Table 1) and the activities of the expressed FtpM with the natural substrate compared to that from the pDK50 FtpM plasmid kindly provided by Prof Hoffmeister. [1] Our N-tagged enzyme resulted in a mixture of truncated and full length protein with lower activity than the C-tagged protein. The pDK50 FtpM plasmid gave lower expression than our C-terminal tagged enzyme. Monomethylation but not dimethylation of the natural substrate 5 was observed with enzyme from expression of all constructs. We also noted a single amino acid difference between our C-terminal tagged enzyme (Lys149) and the original pDK50 enzyme (Glu149) although a AlphaFold 2 model predicts this residue to be remote from the active site and the dimer interface.

C-and N-tagged FtpM
C and N-tagged FtpM was transformed into E. coli SoluBL21 (DE3) cells (Genlantis). A single colony was added to 10 mL LB medium supplemented with 100 μg/mL ampicillin and incubated at 37°C, 120 rpm for 16 h. This was used as a starter culture to inoculate the main culture (1 L LB medium supplemented with 100 μg/mL ampicillin) which was incubated at 37°C, 180 rpm until the culture had reached an OD600 of 0.6-0.8. Gene expression was then induced by the addition of 1 mM IPTG and the temperature was reduced to 16°C and incubated for a further ~ 16 h. The cells were harvested (4000 xg, 10 min, 4°C) and the cell pellet was resuspended in resuspension buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl) and lysed using sonication. Cell debris was removed by centrifugation (15000 xg, 1 h, 4°C) and the supernatant was filtered using a 0.45 μm syringe filter prior to loading onto a HisTrap Fast Flow affinity column (GE Healthcare) equilibrated with resuspension buffer. The column was washed with resuspension buffer containing 50 mM imidazole and protein elution was performed using resuspension buffer containing 500 mM imidazole. Purified protein was buffer exchanged into 50 mM MES pH 6.0 buffer using a PD-10 Sephadex column (GE Healthcare) and protein concentration was increased using a centrifugal filter unit centrifuged at 4000 x g for 10 min. FtpM activity was tested as described in 1.3.1.

pDK50 FtpM
The pDK50 plasmid was provided transformed into E. coli KRX cells. A single colony was added to 10 mL LB medium supplemented with 50 μg/mL kanamycin and incubated at 37°C, 120 rpm for 16 h. This was used as a starter culture to inoculate the main culture (1 L LB medium supplemented with 50 μg/mL kanamycin) which was incubated at 37°C, 180 rpm until the culture had reached an OD600 of 0.35. Temperature was reduced to 16°C and incubated for a further 30 min before gene expression was induced by the addition of 0.1% w/v L-rhamnose and incubated for a further ~ 16 h. The cells were harvested (4000 xg, 10 min, 4°C) and the cell pellet was resuspended in resuspension buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl) and lysed using sonication. Cell debris was removed by centrifugation (15000 xg, 1 h, 4°C) and the supernatant was filtered using a 0.45 μm syringe filter prior to loading onto a HisTrap Fast Flow affinity column (GE Healthcare) equilibrated with resuspension buffer. The column was washed with resuspension buffer containing 50 mM imidazole and protein elution was performed using resuspension buffer containing 500 mM imidazole. Purified protein was buffer exchanged into 80 mM Tris-HCl pH 7.0 buffer supplemented with 5 mM MgCl2 and 100 nm EDTA using a PD-10 Sephadex column (GE Healthcare). FtpM activity was tested as described in 1.3.1.

SAH-nucleosidase
SAH-nucleosidase was transformed into E. coli BL21-gold (DE3) cells (Genlantis). A single colony was added to 10 mL LB medium supplemented with 100 μg/mL ampicillin and incubated at 37°C, 120 rpm for 16 h. This was used as a starter culture to inoculate the main culture (1 L terrific broth autoinduction medium supplemented with 100 μg/mL ampicillin) which was incubated at 37°C, 180 rpm until the culture had reached an OD600 of 0.6-0.8. Gene expression was then induced by the addition of 1 mM IPTG and the temperature was reduced to 18°C and incubated for a further ~ 16 h. The cells were harvested (4000 xg, 10 min, 4°C) and the cell pellet was resuspended in resuspension buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl) and lysed using sonication. Cell debris was removed by centrifugation (15000 xg, 1 h, 4°C) and the supernatant was filtered using a 0.45 μm syringe filter prior to loading onto a HisTrap Fast Flow affinity column (GE Healthcare) equilibrated with resuspension buffer. The column was washed with resuspension buffer containing 50 mM imidazole and protein elution was performed using resuspension buffer containing 500 mM imidazole. Purified protein was buffer exchanged into resuspension buffer using a PD-10 Sephadex column (GE Healthcare). SAH-nuc activity was tested using a colorimetric end-point assay based on an MT assay described by Hendricks et al. [3] 2 μM SAH-nuc was incubated with 1 mM SAH for 1 h at 37°C. 10 μM LuxS was added and incubated for a further 15 min at 37°C. An equal volume of 5 mM Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) was added and the absorbance was measured at 412 nm.

PaoABC
pMN100 was transformed into E. coli TP1000 cells, containing a deletion in the mobAB genes responsible for Moco dinucleotide formation. [2] A single colony was added to 10 mL LB medium supplemented with 100 μg/mL ampicillin and incubated at 37°C, 120 rpm for 16 h. 2 mL of this was used as a starter culture to inoculate the main culture (1 L terrific broth autoinduction medium supplemented with 100 μg/mL ampicillin) which was incubated at 37°C, 120 rpm until the culture had reached an OD600 of 0.6-0.8. Gene expression was then induced by the addition of 20 μM IPTG and 1 mM sodium molybdate was also added to the culture. The temperature was reduced to 22°C and incubated for a further ~ 16 h. The cells were harvested (4000 x g, 10 min, 4°C) and the cell pellet was resuspended in resuspension buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl) and lysed using sonication. Cell debris was removed by centrifugation (15000 xg, 1 h, 4°C) and the supernatant was filtered using a 0.45 μm syringe filter prior to loading onto a HisTrap Fast Flow affinity column (GE Healthcare) equilibrated with resuspension buffer. The column was washed with resuspension buffer containing 10 mM imidazole and protein elution was performed using resuspension buffer containing 333 mM imidazole. Purified protein was buffer exchanged into 50 mM Tris-HCl pH 7.5 supplemented with 1 mM EDTA buffer using a PD-10 Sephadex column (GE Healthcare). PaoABC activity was tested by monitoring the conversion of 1 mM DFF to FDCA by PaoABC at 37°C after 2 h using RP-HPLC using conditions (a) described in 1.3.3.

GOase M3-5
Freeze dried cell lysate was resuspended in resuspension buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl) and filtered using a 0.45 μm syringe filter prior to loading onto a HisTrap Fast Flow affinity column (GE Healthcare) equilibrated with resuspension buffer. The column was washed with resuspension buffer containing 20 mM imidazole and protein elution was performed using resuspension buffer containing 300 mM imidazole. 6 mM CuSO4 was added to the eluent and then buffer exchanged into 100 mM KPi pH 7.0 buffer using a PD-10 Sephadex column (GE Healthcare). PaoABC activity was tested by monitoring the conversion of 1 mM HMF to DFF by GOase M3-5 at 37°C after 1 h using RP-HPLC using conditions (a) described in 1.3.3.

FtpM reaction conditions
FtpM reactions were set up to contain a final concentration of 4 μM SAH-nucleosidase, 2 mM SAM (NEB), 500 μM FtpM and 1 mM substrate in 50 mM MES buffer pH 6.0. Reactions were incubated for 16 hours at 25°C with shaking at 120 rpm. Control reactions were also set up parallel to assay reactions but excluding the addition of FtpM. An equivalent volume of 10% triflouroacetic acid (TFA) was added after reaction incubation and centrifuged at 13000 rpm, 3 min in a table-top centrifuge to precipitate out and remove any protein prior to subsequent analysis.

HMF to FDME cascade conditions
Reactions were set up to contain a final concentration of 1 mM HMF, 1.85 μM GOase M3-5, 3.7 μM PaoABC, 0.17 mg/mL catalase from bovine liver and 0.11 mg/mL horseradish peroxidase in 100 mM KPi buffer pH 7.0. Reactions were incubated for 2 hours at 37°C with shaking at 250 rpm. After 2 hours, the pH was dropped to 6.0 using HCl and 4 μM SAH-nucleosidase, 2 mM SAM (NEB), 500 μM FtpM was added, and reactions were incubated for 16 hours at 25°C with shaking at 120 rpm. An equivalent volume of 10% triflouroacetic acid (TFA) was added after reaction incubation and centrifuged at 13000 rpm, 3 min in a table-top centrifuge to precipitate out and remove any protein prior to subsequent analysis.

RP-HPLC conditions
All Reverse-phase HPLC was performed using an Agilent 1260 Infinity system equipped with a 4.6 x 150 mm ZORBAX Eclipse XDB-C18 5 μM column (Agilent). All standards used for HPLC analysis were either commercially available or synthesised in-house.

HPLC calibration
To fully quantify the final conversion of substrate to product, HPLC peak areas were adjusted using a 1:1 standard of substrate:product(s) or a calibration curve of substrate/product concentration was created.

3.2.3).
To create a calibration curve, 3 standards with 1 mM caffeine as an internal standard and varying concentrations of 0.2 to 1 mM product were run using the above HPLC method (1.3.2). The average product: caffeine peak area ratio was plotted against product concentration to generate the calibration curve. The FtpM reaction was then set up as previously described (1.3.1), with the addition of 1 mM caffeine prior to HPLC injection to fully quantify final concentration of product.
For reactions where a product standard could not be obtained, a calibration curve was created using the above method to quantify the substrate loss to calculate the final conversion to product. This was used to quantify the conversion to product 30 by quantifying the remaining concentration of substrate 29. The same method was also used to quantify the conversion to monoester product 37 by quantifying the concentrations of substrate 36 and diester product 38. S10 Figure S2 Calibration curve used for FDME 3 quantification. Curve was generated as described in section 1.3.5.    Reverse-phase HPLC was performed using an Agilent 1290 Infinity system equipped with a 4.6 x 150 mm ZORBAX Eclipse XDB-C18 column (Agilent). 5 μL of sample was injected and run on a gradient of 0.1% TFA in 5:95 methanol:water to 0.1% TFA in 90:10 methanol: water at a flow rate of 0.6 mL/min for 30 min at 35°C. Masses of product peaks were identified using an Agilent 6540 UHD Accurate Mass Q-TOF.

Kinetics
Reactions were set up to contain a final concentration of 4 μM SAH-nucleosidase, 2 mM SAM (NEB), 100 μM FtpM and varying substrate concentration (0.1-2 mM) in 50 mM MES buffer pH 6.0. Reactions were incubated at 25°C with shaking at 120 rpm. To determine initial rate, samples were taken from the reactions and product formation was monitored via RP-HPLC method (a). The initial rates (nmol min -1 ) were plotted against substrate concentrations (mM) using GraphPad Prism 9 using the Michaelis-Menten model based on non-linear regression to produce a Michaelis-Menten curve and calculate kinetic parameters Vmax and Km. Reactions were performed in triplicate and standard deviation error bars were plotted. Results are shown in section 2.5.1

Synthesis of substrates and product standards 1.4.1 General Synthesis Experimental Details
Unless stated, all materials were purchased from commercial sources (Acros, Aldrich, Alfa Aesar, Fluorochem and Carbosynth) and used without any further treatment. Anhydrous solvents were obtained by passage through drying columns supplied by BBraun Ltd. High-boiling solvents were removed from the reaction crudes employing rotary evaporators connected with high-vacuum pumps. Flash column chromatography was performed using silica gel (Aldrich 40-63 µm, 230-400 mesh). Thin layer chromatography was performed using UV254 sensitive, silica gel coated, aluminium TLC plates purchased from Merck. Visualization was achieved by UV fluorescence or either basic KMnO4 solution or acidic, ethanolic phenol and heat.
All NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer in the deuterated solvent stated. Chemical shifts are reported in ppm and coupling constants (J) are reported in Hz. 1 H NMR spectra were recorded at 500 MHz. 13 C NMR spectra were recorded at 126 MHz and were proton decoupled. Chemical shifts (δ) are given in ppm. Peaks are described as singlets (s), doublets (d), triplets (t), quartets (q), multiplets (m) and broad (br.). Coupling constants (J) are quoted to the nearest 0.5 Hz. All assignments of NMR spectra are based on 2D NMR data (COSY, HSQC-DEPT). IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer as thin films or solids compressed on a diamond plate. Mass spectra were recorded using HRMS were recorded using either an Agilent QTOF 7200 mass spectrometer (CI) or an Agilent 6540A Accurate-Mass Q-ToF MS with Agilent JetStream Source (ESI). Samples were injected using a direct infusion syringe pump. The stated carboxylic acid (1.0 eq.) and amine (1.1 eq.) were suspended in MeCN (3.3 mL/mmol) and EtOAc (0.78 mL/mmol). Pyridine (2.0 eq.) was added and the suspension was cooled to -5 °C. T3P ® (1.5 eq., 50 wt. % in EtOAc) was added dropwise. The resulting suspension was allowed to warm to 0 °C slowly over 3 hours, during which time the reaction mixture became homogeneous. The reaction mixture was diluted with EtOAc (~20 mL/mmol) and washed sequentially with 1 M aq. HCl (~10 mL/mmol), sat. aq. NaHCO3

Synthesis of Natural
(~10 mL/mmol) and sat. aq. NaCl (~10 mL/mmol) then dried over MgSO4 then concentrated in vacuo. The residue was then purified as stated.

General Procedure B: t-Butyl Ester Cleavage Using TFA
The stated t-butyl ester (1.0 eq.) was dissolved in CH2Cl2 (6 mL/mmol) and cooled to 0 °C. TFA (4 mL/mmol) was added dropwise and the reaction mixture was allowed to warm to room temperature and stirred 2 hours. The reaction mixture was concentrated in vacuo and purified, if required, as stated.
This was then left to stir overnight under a nitrogen atmosphere to allow the amide coupling reaction to complete. DCM was removed in vacuo and the residue extracted with ethyl acetate (30 mL x 3) before washing with H2O (30 mL x 2) and brine. The combined organic layers were dried over magnesium sulfate and the solvent was removed in vacuo to obtain the crude product as a brown oil. Purification by column chromatography using a gradient elution of 1% through to 5% MeOH in DCM afforded the title compound S3 as a yellow oil (1.06 g, 66%).
MeOH was removed in vacuo before acidifying to pH 2 with concentrated HCl (1 M). The solution was then extracted with ethyl acetate (20 mL x 3) and washed with H2O (20 mL x 2) and brine (10 mL). The diacid product 5 was obtained was a yellow solid (0.38 g, 40%).

Protein modelling
Protein models of the FtpM monomer and dimer structures were modelled using AlphaFold 2 [7] in its Colab online implementation. [8] The is_training flag was set to True and the num_samples flag to 4 to enable stochastic sampling and increase conformational exploration. The 20 resulting monomer models were all structurally similar and predicted to be of high quality with a mean pLDDT of 89-91 (on a scale of 0-100) and a pTMscore of 0.88 to 0.87 (on a scale of 0-1). Since pLDDT correlates well with model quality [7] this result strongly suggests that the FtpM model was high quality. The top-ranking model was subjected to refinement with AMBER. [9] Twelve dimer models were similarly obtained. The conformation of a single chain was similar in the monomer and dimer models ( Fig  S10) Figure S10 The top-ranking monomer (purple) and dimer (shades of grey) AlphaFold 2 models of FtpM superimposed and visualised in PyMOL (http://pymol.org).
Docked SAM in the monomer model is shown as sticks.
The top-scoring dimer model was used to visualise the position of the termini in order to help interpret the possible implications of alternative locations for His-tags (Fig S11)

Figure S11
The top-ranking dimer model with N-and C-termini marked with red and blue spheres, respectively.
Electrostatic analysis was done with APBS. [10] Small molecule docking was one with Webina 1.0.3 [11] a web implementation of the AutoDock Vina algorithm. [12] Since modelling demonstrated that the substrate binding site lay far from the predicted dimer interface ( Fig S10) the top-ranked FtpM monomer model was used for docking. Small molecule coordinates were obtained either from the Protein Data Bank [13] in the case of SAM (from entry 1ve3) or by using the Molinspiration Cheminformatics server [14] and sketching the molecular structure. PyMOL was used for interconversion of coordinate file formats. SAM was initially placed according to the position of SAH in PDB entry 1m6e: among the FtpM homologues of known structure identified by HHpred [15] , 1m6e was the closest to contain cofactor or cofactor analogue. The initial approximate SAM placement was used to define the centre and size of a box in (extending around 5Å beyond the ligand in each dimension) in which Autodock docked the cofactor. An enhanced level of search rigour was specified by increasing the Exhaustiveness value from the default 4 to 8. Substrate molecules were placed centrally in the easily identifiable substrate-binding and docked similarly. Autodock produced a number of ranked poses and predicted affinities which were examined in PyMOL.      Figure S19 Michaelis-Menten curve used to determine kinetic parameters of the methylation of FMME 10 catalysed by FtpM. Curve was generated as described in section 1.3.7.  [16] Saliyclic acid MT Antirrhinum majus Salicylic acid 0.083 0.66 8 [17] Benzoic acid MT Antirrhinum majus Benzoic acid 1.1 1.2 1.1 [18] Jasmonic acid MT Arabidopsis thaliana Jasmonic acid 0.039 1500 38460 [19] Indole-3-acetic acid MT Arabidopsis thaliana Indole-3-acetic acid 0.013 1.68 129.2 [16]